The transfer of genes into cells provides a means to determine gene function and treat diseases of genetic basis. In addition, gene transfer provides the basis for high-level protein expression, used by molecular researchers to study protein function and to produce new protein drugs. The introduction of genes into animals can also produce useful animal models of human diseases.
Many methods have been developed to introduce exogenous genes into cells. The earliest method for introducing DNA into cells was to incubate the DNA with an inert carbohydrate polymer (dextran) to which a positively charged chemical group (DEAE) had been coupled. These large DNA-containing particles stuck to cells and were taken up by endocytosis. However, this method was inefficient for the introduction of nucleic acid into many types of cells. Another widely used method is the precipitation of DNA with calcium phosphate, which allows transient expression of genes in cells. However, neither of these methods allow the transfer of nucleic acids into a cell with a high efficiency, nor do they allow for persistent gene expression. Thus, a need remains for a high efficiency method for the stable introduction of genes into cells.
Viral vectors, such as adenoviral or retroviral vectors, have been used to introduce foreign DNA with high efficiency. These vectors include adenoviral vectors and retroviral vectors.
The wild type adenovirus genome is approximately 36 kb, of which up to 30 kb can be replaced with foreign DNA. There are four early transcriptional units (E1, E2, E3 and E4), which have regulatory functions, and a late transcript, which codes for structural proteins. Replication-defective vectors have been produced, which have an essential region of the virus (e.g. E1) deleted. Other genes (e.g. E3 or E4) can be also deleted in the replication-deficient vectors. These additional gene deletions increase the capacity of the vector to carry exogenous nucleic acid sequences. The E2 region can also be deleted in a replication-defective vector; this type of vector is known as a “mini Ad,” “gutted vector,” or “gutless vector.” In order to utilize these “gutless vectors”, a helper cell line, (e.g. 293 cells), is needed to provide necessary proteins for virus packaging. Although adenoviral vectors, including gutless vectors, can infect both dividing and non-dividing cells, they generally do not stably integrate into the cellular genome.
In an alternative strategy, retroviruses, such as Moloney murine leukemia virus (MoMLV), have been used to introduce genes into cells. Retroviruses are RNA viruses that, when they infect cells, convert their RNA into a DNA form, which is then integrated into the cellular genome. The integrated provirus can produce RNA from a promoter located in the long terminal repeats (LTRs), which are DNA repeats located at the end of the integrated genome. Retroviral DNA vectors are plasmid DNAs which contain two retroviral LTRs, and a gene of interest inserted in the region internal to these LTRs. The retroviral vector can be packaged by packaging cell lines, containing the gag, pol, and env genes, which provide all the viral proteins required for capsid production and the virion maturation of the vector.
A retroviral vector integrates into the cellular genome once it is introduced into cells, thereby stably transfecting the cells. However, retroviruses can transform only cells that are dividing; retroviral vectors cannot be used to introduce nucleic acid into non-dividing cells.
Recently, hybrid virus expression systems have been developed, which are designed to improve gene transfer protocols where long-term expression is desired in both dividing and non-dividing cells (Duisit et al., Human Gene Therapy 10:189–200, 1999; Caplan et al, Gene Therapy 6:454–459, 1999; Lin, Gene Therapy 5: 1251–1258, 1998). However, since the recombinant vector produced is a retrovirus, cell division is still required for virus infection and integration. This latter fact is a significant drawback to target cells that are terminally differentiated and non-dividing. In addition, each of these systems requires the use of more than one virus. Thus a need remains for a single vector which provides high efficiency, stable transfection in vitro of both dividing and non-dividing cells, without requiring the presence of additional viruses or viral proteins.
The high-efficiency transfer of genes is also of use in vivo. Over the past decade a new approach to the treatment of disease has been developed using genes as therapeutic agents. The goal of gene therapy is to deliver DNA into the body for the treatment of an array of inherited and acquired diseases. Since the first clinical gene therapy protocol for severe combined immunodeficiency disease started in September 1990, more than 300 clinical protocols have been approved worldwide. Clinical experience suggests that gene therapy has the potential to treat a broad range of human diseases, with a low risk of adverse reactions. However, the efficiency of gene transfer and expression in vivo is still relatively low (Donehower, et al., Proc. Natl. Acad. Sci. USA 81: 6461–6465, 1984.
The gene delivery system is frequently the limiting factor for successful gene therapy. Ideally, a gene therapy vector should efficiently and safely deliver therapeutic genes to the target tissues, and should produce a therapeutic amount of gene product for the appropriate time, without requiring any complementing functions supplied in trans. Unfortunately, none of the vector systems presently in use meet all of these requirements.
Both retroviruses (e.g., Moloney Murine Leukemia Virus, MoMLV), and adenoviruses, have been used for human gene therapy. Experience has demonstrated that although a retroviral vector such as a MoMLV vector is a minimal safety risk, its low titer and low gene transfer efficiency make it most suitable for ex vivo use. In addition, as described above, MoMLV can integrate into the genome only in dividing cells.
In contrast to a retrovirus, the transport of an adenovirus to the nucleus is rapid in both dividing and non-dividing cells in vivo. However, an immune response can be generated against the adenoviral proteins produced by an adenoviral vector. In general, the “gutless vectors” induce less of an immune response than other adenoviral vectors. Although adenoviruses can be produced at very high titers and may infect cells with high efficiency, they integrate into the cell genome only at very low frequency, which results in unstable gene expression.
Thus a need exists for a single vector which can be used to stably introduce nucleic acid into both dividing and non-dividing cells, which can be used both in vitro and in vivo.